Abstract

Background

Malate synthase, one of the two enzymes unique to the glyoxylate cycle, is found in
all three domains of life, and is crucial to the utilization of two-carbon compounds
for net biosynthetic pathways such as gluconeogenesis. In addition to the main isoforms
A and G, so named because of their differential expression in E. coli grown on either acetate or glycolate respectively, a third distinct isoform has been
identified. These three isoforms differ considerably in size and sequence conservation.
The A isoform (MSA) comprises ~530 residues, the G isoform (MSG) is ~730 residues,
and this third isoform (MSH-halophilic) is ~430 residues in length. Both isoforms
A and G have been structurally characterized in detail, but no structures have been
reported for the H isoform which has been found thus far only in members of the halophilic
Archaea.

Results

We have solved the structure of a malate synthase H (MSH) isoform member from Haloferax volcanii in complex with glyoxylate at 2.51 Å resolution, and also as a ternary complex with
acetyl-coenzyme A and pyruvate at 1.95 Å. Like the A and G isoforms, MSH is based
on a β8/α8 (TIM) barrel. Unlike previously solved malate synthase structures which
are all monomeric, this enzyme is found in the native state as a trimer/hexamer equilibrium.
Compared to isoforms A and G, MSH displays deletion of an N-terminal domain and a
smaller deletion at the C-terminus. The MSH active site is closely superimposable
with those of MSA and MSG, with the ternary complex indicating a nucleophilic attack
on pyruvate by the enolate intermediate of acetyl-coenzyme A.

Conclusions

The reported structures of MSH from Haloferax volcanii allow a detailed analysis and comparison with previously solved structures of isoforms
A and G. These structural comparisons provide insight into evolutionary relationships
among these isoforms, and also indicate that despite the size and sequence variation,
and the truncated C-terminal domain of the H isoform, the catalytic mechanism is conserved.
Sequence analysis in light of the structure indicates that additional members of isoform
H likely exist in the databases but have been misannotated.